CTHRC1+ fibroblasts and SPP1+ macrophages synergistically contribute to pro-tumorigenic tumor microenvironment in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer that accounts for over 90% of all pancreatic cancer cases. With a 5-year survival rate of only 13%, PDAC has proven to be extremely desmoplastic and immunosuppressive to most current therapies, including chemotherapy and surgical resection. In recent years, focus has shifted to understanding the tumor microenvironment (TME) around PDAC, enabling a greater understanding of biological pathways and intercellular interactions that can ultimately lead to potential for future drug targets. In this study, we leverage a combination of single-cell and spatial transcriptomics to further identify cellular populations and interactions within the highly heterogeneous TME. We demonstrate that SPP1+APOE+ tumor-associated macrophages (TAM) and CTHRC1+GREM1+ cancer-associated myofibroblasts (myCAF) not only act synergistically to promote an immune-suppressive TME through active extracellular matrix (ECM) deposition and epithelial mesenchymal transition (EMT), but are spatially colocalized and correlated, leading to worse prognosis. Our results highlight the crosstalk between stromal and myeloid cells as a significant area of study for future therapeutic targets to treat cancer.

knockout of α-SMA in PDAC has led to tumor indifferentiation, leading to a worse prognosis in both humans and mice 10 , a multi-faceted approach is needed in order to target the stroma.Recently, studies have shed light on the role of collagen triple helix repeat containing 1 (CTHRC1) as a conserved gene regulating pathways associated with fibrosis and cell proliferation, as well as aiding in the transition to myCAF from pancreatic stellate cells 11,12 .Similarly, gremlin-1 (GREM1), a bone morphological protein inhibitor, has been observed in PDAC CAF to increase fibrosis 13 and the infiltration of macrophages in the TME while playing a major role in regulating epithelial cell heterogeneity 14 .In recent years, attention has been increasing on the crosstalk between pro-fibrosis TAM and CAF in particular.
Macrophages play a pivotal role in orchestrating immunosuppression in the TME through diverse mechanisms.They can be generally classified into M1 anti-tumor and M2 pro-tumor 15 , but this system does not show the heterogenic role of TAM in detail.It is widely known that anti-inflammatory and pro-fibrotic M2 TAM secrete various immunosuppressive cytokines and chemokines, along with transforming growth factor-beta (TGF-β) to promote fibrosis, EMT, and foster the recruitment and activation of CAF 16,17 .M2 TAM have also been shown to promote cell proliferation, angiogenesis, and phagocytosis to reduce inflammation 15 .Increased abundance of these TAM are also correlated to enhanced expression of PD-L1 and resistance to anti-PD-L1 treatment, a therapy that has shown to not be effective in PDAC in part due to its non-immunogenic nature 18,19 .Osteopontin (SPP1) has been associated as a hallmark of pro-tumor macrophages 20 , and their functions in the TME have been well established.It is noteworthy that SPP1 + macrophages are implicated in activation of myCAF in other immune mediated diseases, such as kidney and lung fibrosis 21 , as well as directly stimulating CTHRC1 + CAF to deposit ECM components 22 .Apolipoprotein-E (APOE) has also been demostrated to induce immunosuppression 23,24 , supporting a major characteristic of M2 TAM.However, the interactions between SPP1 + TAM and other cells in the TME still requires further investigation.
In this study, we unveil a distinct population of TAM characterized by SPP1 and APOE along with another population of myCAF expressing CTHRC1 and GREM1.Using single cell transcriptomics, we establish a high degree of correlation, highlighting their contributions in immune-suppressive capacities, driving ECM deposition, facilitating matrix remodeling, and EMT, consequently fueling tumor progression.We validate results with spatial transcriptomics, demonstrating the proximity and concordance of these two cellular populations, reinforcing the significance of our results.This research serves as additional insights into the complex crosstalk between macrophage and stromal populations within the PDAC TME.

Results scRNAseq data integration reveals diverse tumor microenvironment
To conduct this study, primary tumor samples (n = 51), metastatic samples (n = 6), and adjacent normal (control) tissue (n = 6) from a small subset of patients were obtained from five public scRNAseq datasets.After filtering for low quality cells or genes, a total population of 98,749 cells and 35,677 genes remained for downstream analysis, revealing an atlas of the TME (Fig. 1a).The major clusters were manually annotated according to differentially expressed (DE) canonical marker genes (Fig. 1b, c): T cells (n = 27,862) were positive for CD3E, CD4, CD8A, and GZMB; B-cells (n = 5071) expressed MS4A1 and CD79A; plasma B (n = 1516) had high expression of CD79A and MZB1; acinar cells (n = 1694) exhibited PRSS1 and REG1A, epithelial cells (n = 29,781) expressed EPCAM and KRT8; endothelial (n = 1803) showed PECAM1 and CDH5; mast cells (n = 2010) were positive for TPSAB1, myeloid cells (n = 21,645) expressed CD16b, CD68, and CD14; and stromal cells (n = 5564) were defined by COL1A1 and LUM.We then combined epithelial with acinar cells, mast cells with myeloid cells, B cells with plasma B cells, and endothelial cells with stromal cells to form five major groups for downstream analysis, with calculated total counts (Fig. 1d) and patient type composition (Fig. 1e), revealing a diverse tumor population composed primarily of epithelial, T-cells, and myeloid cells.These preliminary results show the abundance of immune cells in the TME; however, the poor prognosis and failures of current immunotherapies suggest mechanisms that prevent an effective anti-tumor immune response.

Characterization of T-cells in the PDAC TME
First, we investigated the composition of T cells in the TME, as they are key parts of the adaptive immunity response against tumors.We labeled 11 groups under the general classification of CD4 + /CD8 + T-cells and GZMB + natural killer (NK) cells, and used reported genes in literature to identify specific T cell subtypes (Fig. 2a, b).CD4 + T cells were comprehensively found in greater proportions in tumor populations (Fig. 2c), with CD4 + central memory (Tcm) (Diff = 7.2%, p = 0.031), FOXP3 + T regulatory (Treg) (Diff = 7.6%, p = 0.035), Naive CD4 + (Diff = 13.2%,p = 0.0021), and T helper 2 (Th2) (Diff = 3.6%, p = 0.0059) all enriched.Populations of FOXP3 + Treg have been confirmed mediators in angiogenesis and immune-suppressive functions, contributing to the protumor TME 25 , although a recent study stated the role of Th2 cells in anti-tumorigenic responses 26 .Conversely, the majority of CD8 + T cells were found in lower levels in tumor samples, with activated CD8 + (Diff = 26.7%,p = 0.0026) and CD8 + resident memory (Trm) (Diff = 4.3%, p = 0.0095) being significant in adjacent normal tissue (Fig. 2c).CD8 + effector memory (Tem) (Diff = 8.43%, p = 0.012) was the only CD8 + enriched group in the tumor.Although studies have logically shown the increased presence of exhausted CD8 + T cells (Tex) in tumors due to the immunosuppressive environment 27,28 , we found no significant difference in Tex proportions between healthy adjacent and tumor (Diff = 3.2%, p = 0.088), with even activated CD8 + cells in the tumor surpassing the Tex proportion (Fig. 2c).This suggests that the infiltration and effectiveness of CD8 + T cells in the tumor is extremely low, hence its classification as a "cold tumor" owing to the failures and challenges of anti-PD1/PDL1 therapies in PDAC 29 .The increased number of CD4 + Treg and reduced CD8 + anti-tumor responses indicate the prevalent pro-tumor mechanisms in the TME, and more research is needed to uncover these biological processes. www.nature.com/scientificreports/

Characterization of stromal cells in the TME
As stromal cells have been known to contribute to an immunosuppressive TME while playing a critical role in the production of extracellular matrix in PDAC 30 , we decided to investigate this cell type further to classify specific identities of these populations.Altogether, 12 populations of stromal cells were identified based on previously described gene markers (Fig. 3a, Supplementary Fig. 2A), including PECAM1 + endothelial cells (n = 1803).Smooth muscle cells (n = 1274) and pericytes (n = 523) were classified by high expression of MYL9 and α-SMA (ACTA2), which suggests that α-SMA may not be suitable as a hallmark for myCAF given its high expression across multiple groups (Supplementary Fig. 2B).We therefore distinguished our large subset of myCAF based on their expression of matrix-associated genes (collagens, proteoglycans, and matrix metalloproteinases) and CTHRC1, which were then further divided into two clusters: canonical myCAF (n = 444) with high CTHRC1 expression, and CTHRC1 + GREM1 + myCAF (n = 1413).CLU + fibroblasts (n = 515) resembled a smooth muscle phenotype with expression of CLU and ADIRF.A large population of fibroblasts expressed the complements C3 and C7, suggesting their proinflammatory nature; we differentiated them into three subtypes, C3 + RARRES1 + CAF (n = 873), C3 + SFRP1 + CAF (n = 264), and a group that exhibited both an inflammatory and myofibroblast signature, which we designated C3 + CTHRC1 + CAF (n = 1413).Mesothelial cells acquired high expression of KRT8 and KRT18 (n = 90).A small subset of fibroblasts were shown to be antigen-presenting fibroblasts based on expression of MHC-II and RGS5 (n = 78), while another group expressed MKI67, represented as proliferative CAF (n = 30).Two stromal cell subtypes, CTHRC1 + GREM1 + myCAF (Diff = 23.7%,p = 0.0014) and CLU + CAF (Diff = 5.9%, p = 0.025) were found in significantly higher proportions in tumor patients (Fig. 3b), while other groups such as smooth muscle (Diff = 10.4%),C3 + RARRES1 + CAF (Diff = 9.7%), and canonical myCAF (Diff = 3.5%) were trending towards tumor-enriched.In particular, CTHRC1 + GREM1 + myCAF expressed high levels of collagens (Fig. 3c, Supplementary Fig. 2B, C), including type-1 (COL1A1, COL1A6), type-3 (COL3A1), type-5 (COL5A2), and type-6 (COL6A1), which contribute to tumor cell migration and proliferation through collagen/integrin interactions 31,32 .These fibroblasts also expressed matrix metalloproteinases MMP2, MMP11 and MMP14 (Supplementary Fig. 2B), which are not only key components of matrix remodeling 33 , but lead to cancer progression and worse survival in patients 34,35 .We also observed increased expression of proteoglycans such as CTHRC1 and THBS2 that were unique to myCAF (Fig. 3c, d), which have both been determined to contribute to EMT through the Wnt/b-catenin and NF-kB pathways, leading to tumor invasiveness and poor survival [36][37][38] .In line with these findings, expression of CTHRC1 was upregulated in tumors from all cancers in the TCGA cohort (Supplementary Fig. 2D), suggesting these ECM protein networks are not limited to PDAC.FN1 was also an important gene in CTHRC1 + GREM1 + myCAF (Fig. 3c), which has been shown to promote angiogenesis and metastasis of tumor cells through integrin signaling, leading to the activation of the FAK pathway and also contributing to EMT 39 .Interestingly, ITGB1 and ITGB5 were discovered to be present on these myCAF (Supplementary Fig. 2B), implying the role of these two integrins as a vital form of crosstalk between myCAF and ECM proteins (Supplementary Fig. 2E) 40,41 .
To elucidate the biological pathways of this fibroblast phenotype, gene set enrichment analysis revealed that CTHRC1 + GREM1 + myCAF contributed towards increased levels of TGF-β signaling (Fig. 3d).Further analysis revealed that EMT was an extremely significant mechanism regulated by CTHRC1 + GREM1 + myCAF (Fig. 3e, f, Supplementary Fig. 2F), indicating these fibroblasts support cancerous cell differentiation and proliferation leading to rapid tumor growth and potential metastasis.In addition, these cells were also found to help promote

Characterization of myeloid cells in the TME
Although fibroblasts are the main contributor to ECM deposition and remodeling, the function of myeloid cells as crucial components of the pro-tumorigenic TME in PDAC reveals the need to investigate these complex interactions further 42 .Altogether, we classified our myeloid subset into 15 clusters (Fig. 4a, Supplementary Fig. 3A), including a known population of TPSAB1 + mast cells (n = 2010).Neutrophils were categorized into three groups, with high expression of CD16b and CD62L throughout all cells; the first expressed high levels of interferons (IFIT2 and IFIT3) which were labeled as IFN + neutrophils (n = 1647).Another group expressed high levels of MMP9 and MMP25, which we labeled as MMP9 + neutrophils (n = 356), while the last group was called GMFG + neutrophils (n = 2610).Four subtypes of monocytes were also found, which were labeled as CD14 + CD16 -monocytes (n = 1337), CD16 + monocytes (n = 440), ITGB2 + monocytes (n = 471), and IL1B + monocytes (n = 2880).A main group of classical dendritic cells (cDC) expressed high levels of MHC-II markers and CD74 (n = 1856), and interestingly a small population of plasmacytoid DC were also noted by their expression of CLEC4C and IL3RA

SPP1 + TAM enriched in tumor and contribute to pro-tumorigenic functions and lead to worse survival
We compared proportions of each myeloid subtype in each patient, finding that SPP1 + APOE + TAM (Diff = 9.5%, p = 0.011) and CD62L + IFN-high neutrophils (Diff = 5.0%, p = 0.046) were significantly enriched in primary tumor patients versus adjacent normal (Fig. 4b).SPP1 + VEGFA + TAM were also trending towards increased proportions in tumor samples (Diff = 3.4%, p-value = 0.21).All SPP1 + TAM exhibited the M2-marker CD68 with other marker genes such as CSTB and NUPR1 (Fig. 4C), as well as being enriched for M2 signature under the M1 and M2 classification (Fig. 4d) 43 ; additionally, SPP1 + APOE + TAM exhibited genes associated with ECM remodeling, such as FN1, MMP14, and LGALS1 (Supplementary Fig. 3B) that are also indicative of a pro-fibrotic M2 polarization (Fig. 4d, Supplementary Fig. 3C).As the role of FN1 in ECM signaling and EMT is paramount, we noticed that SPP1 + TAM also exhibited a high EMT signature (Fig. 4e-g), with FN1, LRP1, PLAUR , and TIMP1 highly expressed (Supplementary Fig. 3B) and identified as important components leading to EMT 44,45 .Moreover, pathway analysis revealed that SPP1 + APOE + TAM were regulated by the HIF-1 signaling pathway (Supplementary Fig. 3C, 3D), inducing expression of VEGFA, LDHA, and ALDOA 46,47 , which not only promotes hypoxia (Fig. 4g) but is another approach to induce EMT in tumor cells 48 .We also identified SPP1 + APOE + TAM as major downstream components of mTORC1 signaling (Fig. 4g, Supplementary Fig. 3C), which has important roles in macrophage polarization, tumor metabolism, and protein synthesis 49 , possibly hinting that mTORC1 is a viable candidate for inducing M2 macrophages in PDAC.Survival analysis conducted in the TCGA cohort showed that the top 200 DE gene signature of these TAM led to worse survival for patients (Fig. 4h).Thus, we have established the monumental impact of these macrophages in the immuno-suppresive, pro-fibrotic niche, which we hypothesize may help support the role of CTHRC1 + GREM1 + myCAF and tumor cells further through EMT and other pro-tumor mechanisms.Together, these interactions reveal the multi-faceted roles of SPP1 + TAM in the PDAC TME.

Expressions of CTHRC1 + GREM1 + myCAF and SPP1 + APOE + TAM correspond with worse survival
Because of the significant contributions of CTHCR1 + GREM1 + myCAF and SPP1 + APOE + TAM to a pro-tumorigenic TME, we investigated the potential of a synergistic relationship.To address this hypothesis, we first conducted expression plots of CTHRC1, GREM1, SPP1, and APOE in the TCGA cohort (Fig. 5a, Supplementary Fig. 4A), which were all significantly enriched in tumor samples versus control.To determine if these two groups were related, Spearman correlation (Fig. 5b) revealed a remarkable correlation between the myofibroblast and macrophage signatures (r = 0.87), while the relationship between CTHRC1 and SPP1 alone was also deemed significant (r = 0.25).Survival analysis of CTHRC1 and SPP1 gene expression in PAAD demonstrated that patients with high expression of these genes led to worse survival (Fig. 5c), demonstrating the pro-tumor functions of these genes in the clinical context.Furthermore, the combined gene signature of both groups also resulted in worse prognosis for patients, as well as being enriched in tumor populations (Fig. 5c, d).These results highlight the pro-tumorigenic and positive correlation of CTHRC1 + GREM1 + myCAF and SPP1 + APOE + TAM in the TME in single-cell resolution.Next, we aimed to demonstrate their relationship using spatial transcriptomics, amplifying research significance if found to be consistent with single cell transcriptomics.

Spatial transcriptomics reveals co-localization of CTHRC1 + GREM1 + myCAF and SPP1 + APOE + TAM
To assess cell interactions in the spatial landscape, we conducted spatial transcriptomics on three public datasets acquired from PDAC patients (Fig. 6a, Supplementary Fig. 5A).A large population of ductal cells were identified based on expression of PRSS1 and REG1A; populations of alpha cells (GCG) and beta cells were also observed (INS), but were all categorized under the ductal population.We also identified mixed cellular compartments, including a fibroblast/ductal population characterized by high expression of COL1A1, ACTA2, and REG1A; a fibroblast/malignant population expressed GREM1, TIMP1, and TFF1, and a mixed immune population had markers CD3E and MS4A1.In addition to normal cells of the pancreas, tumor cells were determined based on their expression of EPCAM, CDH1, and ID1, while an epithelial cluster was established based on the unique expression of MUC4, MUC6, and MUC5AC.We also noticed a substantial population of CTHRC1 + GREM1 + myofibroblasts which expressed high levels of ECM genes (CTHRC1, GREM1, FN1, POSTN), aligned with scRNAseq results (Fig. 6b, Supplementary Fig. 5B).Additionally, we determined the aforementioned SPP1 + macrophages, with expression of SPP1, CD68, MARCO, and FN1 (Fig. 6b).We verified this by plotting the CTHRC1 + GREM1 + myCAF, SPP1 + APOE + TAM, and EMT signatures from previous parts of our study (Fig. 6c, Supplementary Fig. 5C), which showed clear overlays, supporting the co-localization of fibroblasts and macrophages while promoting the pro-tumor TME.Furthermore, the myCAF and EMT signature were almost identical, spatially confirming the presence of CTHRC1 + GREM1 + myCAF in supporting EMT.Spearman correlation between the myCAF and TAM signatures (r = 0.64) further confirmed the synergistic relationship between these two groups in the spatial landscape (Fig. 6d).Since all three groups of fibroblasts, macrophages, and tumor cells were identified in close proximity to each other (Fig. 6a), we then quantitatively visualized these spatial localizations with a proximity enrichment heatmap (Fig. 6e), showing that CTHRC1 + GREM1 + myCAF were only in relationship to SPP1 + TAM and tumor cells, while SPP1 + TAM were in close proximity to tumor cells and also to immune cells.

Discovering the intricate crosstalk between CTHRC1 + GREM1 + myCAF, SPP1 + TAM and tumor cells
To understand how these three populations interact, cellular ligand-receptor analysis revealed that SPP1 expressed by macrophages interacted with tumor cells, fibroblasts, and endothelial cells the most (Fig. 6f).SPP1 bound with integrins such as ITGB1 and ITGB5 (Fig. 6g), implying the role of SPP1 in integrin signaling between fibroblasts, macrophages, and tumor cells that lead to EMT and cell-adhesion to the ECM.The SPP1-CD44 axis was also significantly enriched in macrophage crosstalk (Fig. 6g), corresponding to an important pathway for cell surface adhesion and metastasis through the activation of PI3K/Akt and MAPK signaling 50 .SPP1 + macrophages also exemplified high expression of TGFB1 (Supplementary Fig. 5B), suggesting an increase in TGFβ signaling towards tumor cells and fibroblasts that directly contribute towards fibroblast recruitment, cell proliferation and EMT, although TGFβ1 was high across all groups.Similarly, CTHRC1 + GREM1 + myCAF communicated with SPP1 + macrophages and tumor cells through integrin signaling (Supplementary Fig. 5D), where collagen/ integrin pairs and FN1/integrin pairs were prevalent.These interactions not only enhance cell proliferation and transformation 51 , but could serve as a potential factor of polarization and recruitment of macrophages to the TME.Together, these interactions reveal the pathways between fibroblasts, macrophages, and tumor cells that contribute to tumor differentiation, proliferation, and worse prognosis.In line with our previous findings from spatial ligand-receptor analysis, we then conducted an in-silico knockout of three potential osteopontin receptors-CD44, ITGB5, and ITGB1 in the CTHRC1 + GREM1 + myCAF population to validate the impact of SPP1-CD44, SPP1-ITGB5, and SPP1-ITGB1 crosstalks (Fig. 6g, Supplementary Fig. 5D).Given the pro-fibrotic properties of SPP1, we hypothesized observational changes related to ECM associated genes following the virtual KO.Indeed, the top 50 perturbed genes resulting from CD44 KO were closely associated with ECM organization and interaction, including genes such as CTHRC1, FN1, THBS2, SPARC, and various collagens (Fig. 7a).These outcomes suggest that the SPP1-CD44 pair significantly affects the fibrotic functions of myCAF, as the absence of CD44 introduces major perturbations in genes related to fibrosis and ECM.We then repeated our analysis with ITGB5 and ITGB1, with similar results (Fig. 7b, Supplementary Fig. 6A).The loss of ITGB5 affected FN1, MMP11, SDC1, and MMP14, although CTHRC1 was not among the top 50 perturbed genes.ITGB1 KO resulted in changes to CTHRC1, VCAN, and LGALS1.Nevertheless, the convergence of virtual gene perturbations resulting from the knockouts of all three genes underscores the multiple mechanisms of osteopontin secreted by SPP1 + APOE + TAM in inducing fibrotic CTHRC1 + GREM1 + myCAF.

Discussion
Although there have been numerous transcriptomic studies on the TME, much work remains to be done in order to find potential drug targets, including in-depth classifications and characterization of the crosstalk between immune, stroma, and cancer cells.The inadequate therapeutic efficacy of current immunotherapeutic clinical trials underscore the importance of finding methods to overcome the immunosuppressive TME 52 .Through further analysis of single-cell RNA-seq data and using the latest approaches in spatial transcriptomics, we highlight the spatial proximity of SPP1 + APOE + TAM and CTHRC1 + GREM1 + myCAF that concordantly contribute to the establishment of a pro-tumor TME involving immuno-regulatory, pro-fibrotic, EMT, and other mechanisms.These types of cellular compartments contain potential as cellular targets for future immune therapies.
In this study, we found that CTHRC1 + GREM1 + myCAF were enriched in tumor patients and expressed significantly higher levels of matrix-associated genes such as proteoglycans (ACTB, DCN), collagens (COL1A2, COL3A1), and matrix metalloproteinases (MMP2, MMP11, MMP14), indicating fibrotic properties and contributing to worse overall survival.To note, we did not conduct trajectory analysis and investigate the potential lineage of myCAF; however, studies done by Jin et al. 53 and Kang et al. 12 have revealed the function of CTHRC1 in activating pancreatic stellate cells (PSCs), which are responsible for the differentiation of PSCs into myCAF,   54 discovered a state of SFRP1 + transitional fibroblasts preceding CTHRC1 + myCAF, which we identified in a small population of our stromal cells.Acknowledging the α-SMA +/− classification of myCAF activation, our analysis revealed that α-SMA was not a defining gene of our myCAF population, and we suggest that CTHRC1 may serve as a potential future marker for myCAF in PDAC and other solid tumor cancers due to its increased expression and positive contribution to the carcinogenesis of other cancers, such as stomach, liver, colon, and breast 11,55 .Jorgensen et al. 56 also defined pro-or-anti-tumor fibroblasts in the pancreas based on CD105 classification.However, CD105 was not highly expressed in CTHRC1 + GREM1 + myCAF, thus it was not a significant defining factor given the heterogeneity of CAF populations among different patients.Future studies will need to be conducted not only in PDAC, but in other cancers to validate the potential of CTHRC1 as a defining marker in myCAF.
Recent research has been conducted to investigate whether targeting myCAF can lead to better outcomes.Nevertheless, reducing myCAF by targeting α-SMA in the tumor led to reduced survival rates in both humans and mice, due to the duality of extensive ECM protein deposition as an immune-physical barrier, restricting immune cell accessibility to cancer cells, but simultaneously restricting tumor growth to an extent 10 .However, our results highlight other α-SMA + populations such as pericytes and smooth muscle cells that potentially could have been inadvertently targeted in their study.These results emphasize the need to thoroughly characterize and target specific fibroblast populations in the TME as therapeutic targets.Here, the next focus of our study looks into the roles of SPP1 and SPP1 + macrophages.We not only identified two major groups of SPP1 + macrophages that are anti-inflammatory M2 and pro-fibrotic like, but they also contributed to hypoxia and EMT, aiding CTHRC1 + GREM1 + myCAF in a synergistic relationship to promote ECM deposition through SPP1-CD44, SPP1-ITGB5, and SPP1-ITGB1 interactions.Recent studies have indicated SPP1 in cancers as a marker of pro-tumor macrophages and cancer cells, leading to worse prognosis for patients 51 .Moreover, Eun et al. 57 demonstrated the role of SPP1 as a prognostic marker in hepatocellular carcinoma fibroblasts, leading to direct chemoresistance against tyrosine kinase inhibitors while driving EMT.Most importantly, the multifunctional presence of SPP1 not only promotes stimulation of myCAF 22 , but also drives EMT and chemoresistance to anti-PDL1 therapy through the PI3K/AKt-mTOR pathway 50,58 .
In-vivo studies have highlighted how SPP1 − knockout mice have been shown to lead to decreased M0/M2 infiltration, a decrease in fibrosis, and exhibit positive results in OS 59,60 ; despite these encouraging results, no current human clinical studies inhibiting SPP1 have been conducted on cancer.We found that the pro-fibrotic SPP1 + matrisome-associated macrophages (MAM) described by Ouyang et al. 61 corresponded with the signature of SPP1 + APOE + macrophages in this study, confirming the role of SPP1 + macrophages in ECM development.Moreover, Fabre et al. 21characterized a broad population of SPP1 + CD9 + TREM2 + macrophages in liver and lung fibrotic diseases, while demonstrating the roles of type-3 inflammation cytokines GM-CSF and IL-17A in inducing these macrophages in-vitro and in-vivo.Importantly, this population matched our scRNAseq SPP1 + TAM signature (SPP1, GPNMB, CD63, FABP5) precisely, suggesting that the presence and functions of their TAM can also be applied to PDAC.Our SPP1 + TAM population also expressed CD68 but had low expression of CD206 and CD163, which differed slightly from another study of SPP1 + macrophages in colorectal cancer that had high CD206 expression 62 .Altogether, these studies highlight the role of SPP1 + TAM in pro-tumor fibrosis; we further describe the functions of these cells in regard to their relationship with fibroblasts and tumor cells.
To date, there have been a few pieces of literature that have investigated the relationship between SPP1 + macrophages and myCAF; however, the emphasis on a positive correlation is unmistakably clear.Qi et al. 62 first did an in-depth study on the network between FAP + CAF and SPP1 + TAM in colorectal cancer, proving the synergistic and spatial correlations of these groups, although their characterization of SPP1 + TAM in the study suggested a M1 pro-inflammatory phenotype regulated by STAT1.CXCL4 was also investigated as a driving factor behind the activation of SPP1 + TAM, concurrently leading to activation of myCAF 63 .Most importantly, Liu et al. 22 directly demonstrated the role of SPP1 in driving collagen deposition in CTHRC1 + myofibroblasts.Our results extend these implications to PDAC, highlighting the importance of the spatially correlated crosstalk between CTHRC1 + GREM1 + myCAF and SPP1 + APOE + TAM that drive fibrosis, immunosuppression, and EMT in PDAC and potentially other cancers.However, more work is needed to describe in particular how these groups interact with T-cells and other anti-tumor responses.Considering the significance of these results in furthering our understanding of macrophage-fibroblast communication, these populations serve as potential therapeutic targets for not only PDAC, but applicable in a wide range of cancers and fibrotic diseases.

Data collection
Altogether, all data analyzed in this study were acquired from public datasets, and secondary analysis was conducted using available data.No new data or reagents were generated with this study.Five single-cell RNAseq datasets were downloaded from the Gene Expression Omnibus (GSE242230, GSE154778, GSE155698, GSE205354, GSE212966), and one spatial dataset (GSE211895) were acquired for analysis.63 patients were in this study, including adjacent normal (n = 6), primary PDAC (n = 51), and metastasis (n = 6), who were in various stages of cancer progression (mostly stage 3 or 4, or taken after patient mortality).

Preprocessing, dimension reduction and clustering
All analysis was conducted in the jupyter notebook software using scanpy python package.Low quality cells and/ or genes were filtered by gene counts and gene expression with the following restrictions: (1) cells less than 500 total gene counts, (2) genes found in less than three cells, (3) cells with over 100,000 total counts.Doublet removal was not performed during this step and subsequently removed manually when annotating clusters.Cell counts

Figure 1 .
Figure 1.Canonical cell markers in PDAC TME.(a) Uniform Manifold Approximation Projection (UMAP) of the comprehensive tumor microenvironment, with a total of 63 samples, 98,749 cells and 35,677 genes.(b) Dot plot of canonical cell marker genes used to annotate major cells.(c) UMAP of canonical marker genes.(d) Bar plot of number of cells per cell type.(e) Bar plot of percentages of each condition per cell type.